Memory T-cell enriched haploidentical transplantation with NK cell addback results in promising long-term outcomes: a phase II trial

Background Relapse remains a challenge after transplantation in pediatric patients with hematological malignancies. Myeloablative regimens used for disease control are associated with acute and long-term adverse effects. We used a CD45RA-depleted haploidentical graft for adoptive transfer of memory T cells combined with NK-cell addback and hypothesized that maximizing the graft-versus-leukemia (GVL) effect might allow for reduction in intensity of conditioning regimen. Methods In this phase II clinical trial (NCT01807611), 72 patients with hematological malignancies (complete remission (CR)1: 25, ≥ CR2: 28, refractory disease: 19) received haploidentical CD34 + enriched and CD45RA-depleted hematopoietic progenitor cell grafts followed by NK-cell infusion. Conditioning included fludarabine, thiotepa, melphalan, cyclophosphamide, total lymphoid irradiation, and graft-versus-host disease (GVHD) prophylaxis consisted of a short-course sirolimus or mycophenolate mofetil without serotherapy. Results The 3-year overall survival (OS) and event-free-survival (EFS) for patients in CR1 were 92% (95% CI:72–98) and 88% (95% CI: 67–96); ≥ CR2 were 81% (95% CI: 61–92) and 68% (95% CI: 47–82) and refractory disease were 32% (95% CI: 11–54) and 20% (95% CI: 6–40). The 3-year EFS for all patients in morphological CR was 77% (95% CI: 64–87) with no difference amongst recipients with or without minimal residual disease (P = 0.2992). Immune reconstitution was rapid, with mean CD3 and CD4 T-cell counts of 410/μL and 140/μL at day + 30. Cumulative incidence of acute GVHD and chronic GVHD was 36% and 26% but most patients with acute GVHD recovered rapidly with therapy. Lower rates of grade III-IV acute GVHD were observed with NK-cell alloreactive donors (P = 0.004), and higher rates of moderate/severe chronic GVHD occurred with maternal donors (P = 0.035). Conclusion The combination of a CD45RA-depleted graft and NK-cell addback led to robust immune reconstitution maximizing the GVL effect and allowed for use of a submyeloablative, TBI-free conditioning regimen that was associated with excellent EFS resulting in promising long-term outcomes in this high-risk population. The trial is registered at ClinicalTrials.gov (NCT01807611). Supplementary Information The online version contains supplementary material available at 10.1186/s13045-024-01567-0.


Introduction
Allogeneic hematopoietic cell transplantation (HCT) can be curative for patients with high-risk hematological malignancies.This therapeutic benefit is mediated by the cytotoxic conditioning regimen and the graftversus-leukemia (GVL) effect.Reducing the intensity of conditioning regimens and specifically avoiding total body irradiation (TBI), particularly in pediatric patients, can significantly lower the incidence of adverse effects, but is associated with higher relapse rates [1][2][3][4][5].
We therefore developed a transplant protocol that consisted of a TBI-free, serotherapy-free, sub-myeloablative conditioning regimen followed by the sequential infusion of haploidentical CD34-enriched and CD45RA-depleted hematopoietic progenitor cell (HPC) grafts combined with an NK-cell addback derived from preferentially NK alloreactive donors.Here we report the outcomes of our prospective clinical trial using this approach in 72 pediatric patients with high-risk hematologic malignancies.

Patients and donors
Written informed consent was obtained in accordance with the Declaration of Helsinki.The study was approved by St. Jude's Institutional Review Board and Investigational Device Exemption for the use of the CliniMACS Plus device (Miltenyi Biotec) was granted by the Food and Drug Administration.The trial is registered at ClinicalTrials.gov(NCT01807611).
Patients with high-risk hematologic malignancy for which HCT was indicated but who lacked a suitable Human Leukocyte Antigen (HLA) matched donor were eligible.Complete remission (CR) was defined as absence of morphological disease and included patients with evidence of minimal residual disease by flow cytometry (MRD: < 0.1% for AML, < 0.01% for ALL) and those with detectible disease (evidence of disease including positive cytogenetic, molecular or flow-cytometric testing but less than as defined above by flow MRD).Active disease was defined as > 5% blasts by morphology prior to transplant, or aplasia with the immediate prior bone marrow evaluation showing > 5% blasts by morphology.Detailed inclusion and exclusion criteria are listed in the provided protocol.
Haploidentical family donors were selected based on established criteria taking donor age, parity, Cytomegalovirus (CMV) status and blood type into account.Donor KIR profiling was also performed [30,31].Initially only patients with a KIR receptor-ligand mismatched haploidentical donor were eligible; subsequently this criterion was waived.

Transplant regimen
The preparative regimen consisted of 8 Gy total-lymphoid irradiation (TLI; four equal fractions), 30 mg/m 2 fludarabine × 5 days, cyclophosphamide 60 mg/kg × 1 day, thiotepa 5 mg/kg × 2 on 1 day, and melphalan 70 mg/ m 2 × 2 days.The preparative regimen was based on previous institutional protocols using fludarabine, thiotepa and melphalan.As serotherapy was being omitted, cyclophosphamide was included for additional immune modulation and TLI was included to promote immunological tolerance and decrease risk of rejection.Patients received a G-CSF-mobilized CD34 + enriched HPC graft on day 0, and a second, CD45RA-depleted HPC graft on day 0/ + 1.On day + 6, they received a purified NK-cell infusion [32] from the same donor.G-CSF was started on day + 7. Sirolimus (n = 9) or mycophenolate mofetil (n = 63) was started 1 week following NK-cell infusion to stop before day + 60 (Fig. 1A).

Correlative studies
Flow cytometry quantification of lymphocyte subsets, T-cell receptor excision circle (TREC), T-cell receptor (TCR) diversity by Vβ spectratyping, and enzyme-linked immunospot (Elispot) assays were performed on subsets of patients.
Details for transplant approaches and correlative studies are provided in Additional file 1: Supplementary Methods.

Statistical analysis
This was a phase II clinical trial designed with Simon's 2-stage optimum design [33] and an expansion cohort.The study included stopping rules for excessive rates of acute GVHD (aGVHD) and non-relapse mortality (NRM).The aGVHD stopping rule was triggered following accrual of 72 of the planned 75 subjects.This manuscript summarizes data as of April 2022 for the 'full cohort' and the subset of these patients in complete morphologic remission at the time of HCT as the 'CR cohort' .The statistical analysis approach is described in Additional file 1: Supplementary Methods.

Patient and donor characteristics
From June 6, 2013, to November 20, 2019, 81 pediatric patients with hematological malignancies were enrolled.
The outcome of subsets of patients were previously reported [34][35][36].Three patients did not proceed to transplant.Six patients received maraviroc for GVHD prophylaxis and were not included in the analysis (Additional file 1: Supplementary Methods, Supplementary Table 1).Seventy-two patients received protocol-specified therapy and are the focus of these analyses.Of the 72 evaluable patients (Table 1), 58% were males and the median age at transplant was 8 years (range: 0.6-20.8).Nineteen patients had active disease at time of transplantation and 53 patients were in morphologic CR (CR1: 25, ≥ CR2: 28).Amongst patients in morphologic CR, 20 (37.7%) had detectable disease, including 9 who were MRD-positive by flow cytometry.Of the donors, 38 were male with the majority being a parent.Most were mismatched at 4/8 HLA alleles, and 62 (86%) were NK alloreactive as defined by KIR receptor-ligand mismatch.

Graft composition, engraftment and chimerism
After receiving protocol defined conditioning and both HPC grafts, 62 patients received NK-cell infusion on day + 6.Ten patients did not receive NK cells due to high-grade fevers and/or other complications.Figure 1B depicts HPC graft doses and Additional file 1: Supplementary Tables 2A, B summarize graft selection and depletion efficiencies.Seventy (97.2%) patients engrafted with a median time to neutrophil engraftment of 11 (9)(10)(11)(12)(13) days and platelet engraftment to > 50,000/μL of 17 (10-98) days (Fig. 1C).One patient experienced acute graft rejection, and another died prior to engraftment.Post-infusion, patients developed an inflammatory syndrome consisting of fevers and tachycardia which correlated with elevated serum C-reactive protein (CRP) levels that peaked at day + 8 (Fig. 1D).Thirty-two (44%) patients met CTCAE v3 criteria for engraftment/cytokine release/macrophage  3).All engrafted patients had 100% donor chimerism at first chimerism evaluation performed soon after neutrophil engraftment, except 2 with 99% donor chimerism.Donor chimerism was 100% at day + 100 and at 1-year in all assessed patients, although several patients had intermittent mixed chimerism.

Viral reactivation
Twenty-five patients developed CMV DNAemia with a cumulative incidence of 36.1% and 5 patients developed adenovirus (Adv) DNAemia with a cumulative incidence of 8.3% at 6 months.There were no cases of Epstein Barr Virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD).Details of infections are listed in Additional file 1: Supplementary Table 3.

Immune reconstitution
At 1-month post-transplant, the median CD3, CD4 and CD8 T-cell counts were 410/μL,140/μL and 200/μL (Fig. 1E).NK-and B-cell counts reached normal values at 1-and 3-months post-transplant (Fig. 1E).Of note, T-cell chimerism was not separately analyzed.For the first 6-months post-transplant, reconstituting T cells had a predominantly memory phenotype (Fig. 1F,G) and a diverse TCR repertoire (Fig. 1H), recapitulating graft content and highlighting the contribution of the infused memory T cells to the rapid immune reconstitution.Starting 3-months post-transplant, naïve T cells emerged, as confirmed by TREC analysis (Fig. 1I).Elispot assays revealed functional immune reconstitution as early as 1-month post-transplant (Fig. 1J) with detection of CMV-or Adv-specific T cells in patients with CMV reactivation or Adv-positive stool and/or blood samples (Fig. 1K).The presence of CMV-and Adv-specific T cells was confirmed in donors (Fig. 1L).
There was however no difference in OS and EFS amongst patients transplanted with or without positive flow MRD (P = 0.4157 and P = 0.2992).
Univariate analyses for all patients (full cohort) and for patients in morphological CR (CR cohort) were performed.For the full cohort, disease status (CR) was associated with better OS (P < 0.0001) and higher graft CD3 T cell dose with worse OS (P = 0.046).In the CR cohort, cGVHD was associated with lower OS (P = 0.077) (Additional file 1: Supplementary Table 6).On univariate analysis for EFS in the full cohort, disease status (CR) and CMV serostatus (CMV-seronegative recipient) were associated with better EFS (P < 0.0001, P = 0.0514) and in the CR cohort, CMV serostatus (CMV-seronegative recipient) and increasing number of HLA mismatches (MM) were associated with better EFS (P = 0.0454, P = 0.0594) (Table 2).In multivariate analysis for OS and EFS, only disease status for the full cohort remained significant (P = 0.0004) (Table 3).
Univariate analysis for the full cohort showed being in CR and being a CMV-seronegative recipient regardless of CMV-donor status were significantly associated with lower relapse risk (P = 0.0012, P = 0.0357).In the CR cohort, CR1, recipient CMV seronegativity, and increasing number of HLA mismatches were significant for lower risk of relapse (P = 0.0114, P = 0.0172, P = 0.0236) (Table 2).On multivariate analysis, only disease status remained significant (P = 0.0330) for the full cohort.In the CR cohort, recipients that were CMV-seronegative had the lowest rate of relapse (0/17) (P = 0.0347) but as 'zero' patients experienced relapse, additional statistical modeling needed to be performed (Additional file 1: Supplementary Methods) (Table 3).

Cumulative incidence of Non-Relapse Mortality
The 3-year cumulative incidence of NRM for the entire cohort was 11.5% (95% CI, 5.2-20.3)(Fig. 2H, Additional file 1: Supplementary Table 5).The incidence of NRM was higher in patients with active disease (5/19) compared to patients in morphologic CR (5/53), and 6/10 deaths were in patients with AML.Four deaths occurred early (< 100 days) due to organ toxicities, and   Excludes 1 patient with active disease at the time of HCT, as they died prior to engraftment and not at risk of relapse of the 6 deaths that occurred > 100 days, 4 were attributable to cGVHD and 1 patient had recently completed therapy for cGVHD.On univariate analysis, in the full cohort, having female donor, highest tertile CD3 count in the infused graft, and occurrence of cGVHD were associated with worse NRM; in the CR cohort, worse NRM was associated with having a female donor and presence of cGVHD (Table 2, Additional file 1: Supplementary Table 6).On multivariate analysis, high CD3 + dose in the graft and maternal donor remained significant for worse NRM in the full cohort.Only maternal donor was significant for worse NRM for the CR cohort (Table 3).

Discussion
In this prospective clinical trial, pediatric patients with high-risk hematological malignancies received a submyeloablative, TBI-and serotherapy-free conditioning regimen followed by CD34-enriched and CD45RAdepleted haploidentical donor grafts and NK-cell addback.This regimen was associated with rapid, robust immune reconstitution and low rates of graft rejection, viral reactivation, and relapse.The EFS using our approach compares favorably to other transplant strategies using matched donors, despite the inclusion of patients with MRD and while using a sub-myeloablative, TBI free approach.While the incidence of GVHD was higher than observed with more stringent T-cell depleted grafts, most patients recovered rapidly with therapy, resulting in promising long-term outcomes in this high-risk population.Importantly, modifiable factors were identified to mitigate the risk of developing GVHD in subsequent trials.Finally, as these results were obtained using haploidentical donors for patients with no other suitable donor choices, our strategy could provide excellent outcomes for all pediatric patients in need of an HCT.Despite using an ex-vivo T-cell depletion strategy, the avoidance of serotherapy along with the large number of infused CD34 + and CD45RA-depleted cells led to rapid immune reconstitution with mean CD3 and CD4 T-cell counts of 400 cells/μL and 140 cells/μL at day + 30.These T cells had a predominantly memory phenotype suggesting adoptive transfer from the infused graft.In contrast, other T-cell depletion approaches such as TCRαβ depletion, achieve comparable T-cell counts only at month 6 post-transplant [37].The early reconstitution is clinically relevant as CD4 counts of ≥ 50 cells/μL at day + 100 are associated with improved survival [38,39].
This robust immune reconstitution translated to low rates of viral infection.The cumulative incidence of CMV DNAemia (36.1%) compares favorably to other studies, including for recipients of T-replete grafts [40][41][42].The cumulative incidence of Adv DNAemia (8.3%) was lower than in other pediatric studies that report incidence of 10-26% and a mortality rate of up to 50% [43][44][45][46][47].Despite the use of a T-deplete graft the incidence of EBV-PTLD was 0% without the use of prophylactic Rituximab or CD19-depletion of the graft.This suggests that the infused donor memory T cells not only reconstituted rapidly but retained their ability to respond to viral infections, a conclusion corroborated by our Elispot results demonstrating functional virus-specific T cells as early as 1-month post-transplant.
The 3-year EFS for patients in morphologic CR including those with MRD positive and detectable disease was 77.4% and for those with flow MRD-negative CR was 85.2%.This compares favorably to other T-cell depletion approaches, including TCRαβ or CD45RA-depletion [37,48,49] despite the use of a sub-myeloablative TBI-free conditioning regimen in our cohort.In contrast, other studies including a large trial using TCRαβ depletion and the recent randomized FORUM trial, show that the use of TBI is required to maintain low relapse rates for patients with ALL [37,48,50].Further, patients with or without pre-HCT MRD positivity in our cohort had no difference in EFS despite several prior studies showing negative impact of pre-HCT MRD on EFS [37,[51][52][53].Our outcome suggests that robust reconstitution with multiple immune effectors provides a potent GVL effect and enables the use of sub-myeloablative, TBI-free conditioning regimens without compromising EFS in our highrisk population.
Active disease (> 5% blasts) at time of transplant and decreasing number of HLA mismatches were associated with higher rates of relapse, with the latter suggesting that the GVL effect could be enhanced by selecting a full haplotype mismatched donor.Intriguingly, CMV-seronegative recipients had low rates of relapse.Most previous studies have evaluated effect of CMV reactivation (not serostatus) on leukemic relapse [54][55][56][57] and whether or not there is a protective effect remains controversial [54,55,58].However, one large study evaluated serostatus and showed higher relapse rates in patients transplanted with CMV-seropositive matched unrelated donors [41].The mechanism of protection from relapse in CMVseronegative recipients in our study remains elusive and warrants further exploration particularly in the context of early NK-cell addback.
Compared to more stringent T-cell depletion approaches, the rates of aGVHD and cGVHD were higher in our study.This could be due to the very high absolute number of T cells infused in the graft.In clinical trials, the median number of haploidentical T cells infused with the use of TCRαβ depletion is 10 4 T cells/ kg [48,59] and with CD45RA-depletion in the matched related and unrelated donor setting, is 10 7 T cells/kg [49].We infused a median haploidentical T-cell dose of 6.08 × 10 7 /kg.The predominant memory T-cell content in the infused graft likely made large doses of infused haploidentical T cells tolerable as > 10 5 unmanipulated haploidentical T cells/kg cause GVHD.As reported by others [49], aGVHD in our study was responsive to therapy and required systemic steroids for a median of < 2 months, confirming preclinical studies that have highlighted differences between aGVHD induced by naïve and memory T cells [60,61].In addition, we used a short course of sirolimus or mycophenolate mofetil as GVHD prophylaxis.However, other approaches could be explored in the future such as the use of abatacept, which has the added advantage of preserving NK-cell function [62][63][64].
The cumulative incidence of aGVHD was substantially higher in patients who received transplants from non-NK-cell alloreactive compared to NK-cell alloreactive donors (70% vs 22.6%).Intriguingly, the impact was differentially increased with increasing number of KIR mismatches.Altogether, we found that NK-alloreactive donor, increasing KIR mismatches and lower donor KIR B haplotype content, were each associated with significantly lower rates of grade III/IV aGVHD but not cGVHD.Pre-clinical and clinical studies have shown that high numbers of alloreactive NK cells can decrease the occurrence of aGVHD [11,[65][66][67] by depleting antigen-presenting cells [11,68] or activated T cells [69,70], and by controlling autologous T-cell responses [71,72], potentially explaining our findings.Similar clinical findings have been reported by other groups that utilized more stringent T-cell depletion techniques [11].However, the number of recipients of transplants from non-NK-cell alloreactive donors in our study was small, and future studies are needed to explore the relationship between NK-cell alloreactivity and aGVHD in larger cohorts receiving CD45RA-depleted transplants.
The use of maternal donors in our study was associated with development of cGVHD but not aGVHD.There have been conflicting reports with occurrence of cGVHD and use of maternal donors and while others have reported similar findings [42,73,74], the relationship between maternal donors and GVHD is dependent on transplant platform utilized [42] and in our context could be due to sensitization of maternal donors to unshared HLA during pregnancies with memory T cells retained in the infused graft.While not statistically significant, a higher CD3 cell dose in the graft was associated with higher rates of cGVHD.
NRM in our cohort, particularly for patients in CR was low (5.7%) and compares favorably with other pediatric transplant studies [42,51,75].The majority of NRM occurred in patients with active disease, where high CD3 cell dose in the graft was associated with early NRM due to organ toxicities.In patients in CR, NRM occurred late and was associated with mother as donor, which was related to development of cGVHD.
There are some limitations to our approach.The cost and complexity of ex vivo graft manipulation in its current form may limit its implementation to centers.The apheresis procedures required in our study were more extensive than for standard transplantation but were well tolerated and feasible as majority of donors were parents or family members who were highly motivated.Modifications to simplify the process to one cell infusion of a CD45RA-depleted product [76], instead of two infusions will make our approach more accessible.Likewise, the use of fully automated, closed system selection devices hold the promise of simplifying the process.While other haploidentical transplant approaches such as use of T-replete grafts and post-transplant cyclophosphamide are easier and less expensive, only ex vivo graft manipulation enables the selective infusion of immune effector cell subsets to study their contribution to transplant outcomes.We believe that studies like ours are critical to develop approaches to enhance GVL effects in the setting of reduced toxicity conditioning regimens to achieve comparable outcomes to standard transplant platforms with the ultimate goal to separate GVL effects from GVHD with minimal exposure to toxic chemo-radiotherapies.
In summary, our study using multiple immune effectors derived from haploidentical donors following a submyeloablative, non-TBI based regimen was associated with excellent outcomes in pediatric patients with highrisk hematological malignancies.The use of NK-alloreactive donors, avoiding maternal donors, and lowering the infused CD3 dose has the potential to mitigate the risk of GVHD in future studies.In addition, understanding the biological mechanism of relapse protection in CMVseronegative recipients could lead to further refinement of our approach.

Fig. 2
Fig. 2 Relapse and survival.A Probability of OS and EFS for the full cohort.B Probability of OS and EFS for CR cohort.C Probability of OS and EFS for patients with active disease.D Probability of OS for patients in CR1, ≥ CR2.E Probability of EFS for patients in CR1, ≥ CR2.F Probability of OS for patients with non-detectible and detectible disease at time of transplant.G Probability of EFS for patients with non-detectible and detectible disease at time of transplant.(Fig F,G,-Six patients excluded due to unknown minimal residual disease (MRD) status) H Cumulative incidence of relapse and NRM for full cohort.I Cumulative incidence of relapse for patients in CR1, ≥ CR2, active disease (1 patient with active disease excluded due to death prior to engraftment).OS, overall survival; EFS, event-free survival; NRM, non-relapse mortality; CR, complete remission, CIR, cumulative incidence of relapse.95% CI is listed in parenthesis

Fig. 3
Fig. 3 GVHD and survival.A Cumulative incidence of all aGVHD and grade III-IV aGVHD.B Cumulative incidence of all cGVHD and moderate-severe cGVHD.C Probability of OS from onset of grade III-IV aGVHD.D Probability of OS from onset of cGVHD.aGVHD, acute graft-versus-host disease; cGVHD, chronic graft-versus-host disease, OS, overall survival.95% CI is listed in parenthesis

Table 2
Selected univariable analysis results*

Table 3
Multivariable model results* * Excludes 1 patient with active disease at the time of HCT, as they died prior to engraftment and not at risk of relapse CR, Complete Remission; aGVHD; acute graft versus host disease; cGVHD, chronic graft vesus host disease; CMV, Cytomegalovirus; R, Recipient; NK, Natural Killer; HLA, Human Leukocyte Antigen; MM, mismatch; KIR, Killer Immunoglobulin Receptor; rec-lig, receptor-ligand * Model specifications differ by cohort and by outcome (details in the Additional file 1: Supplementary Materials); two-sided P-values are reported *